Andrew O. Jo

TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4−/− and Aqp4−/− mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca2+]i elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca2+]i elevations but only modestly attenuated the amplitude of Ca2+ signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca2+ entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4–AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. SIGNIFICANCE STATEMENT We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation.

Swelling and Eicosanoid Metabolites Differentially Gate TRPV4 Channels in Retinal Neurons and Glia

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca2+]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4−/− Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+ waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+ signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function.

TRPV1 and Endocannabinoids: Emerging Molecular Signals that Modulate Mammalian Vision

Transient Receptor Potential Vanilloid 1 (TRPV1) subunits form a polymodal cation channel responsive to capsaicin, heat, acidity and endogenous metabolites of polyunsaturated fatty acids. While originally reported to serve as a pain and heat detector in the peripheral nervous system, TRPV1 has been implicated in the modulation of blood flow and osmoregulation but also neurotransmission, postsynaptic neuronal excitability and synaptic plasticity within the central nervous system. In addition to its central role in nociception, evidence is accumulating that TRPV1 contributes to stimulus transduction and/or processing in other sensory modalities, including thermosensation, mechanotransduction and vision. For example, TRPV1, in conjunction with intrinsic cannabinoid signaling, might contribute to retinal ganglion cell (RGC) axonal transport and excitability, cytokine release from microglial cells and regulation of retinal vasculature. While excessive TRPV1 activity was proposed to induce RGC excitotoxicity, physiological TRPV1 activity might serve a neuroprotective function within the complex context of retinal endocannabinoid signaling. In this review we evaluate the current evidence for localization and function of TRPV1 channels within the mammalian retina and explore the potential interaction of this intriguing nociceptor with endogenous agonists and modulators.

Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels

Significance The secretion of aqueous humor from the ciliary body is regulated by osmotic gradients, yet the mechanism through which these cells sense these gradients is still under debate. We have identified the calcium-permeable transient receptor potential vanilloid isoform 4 (TRPV4) ion channel as critical for translating hypotonic stimuli into intracellular signals and linked the activation of this channel to a known proinflammatory lipid signaling pathway. The channel was confined to nonpigmented cells that secrete aqueous fluid and regulate intraocular pressure. Thus, activation of TRPV4 may contribute to vision through metabolic support of anterior eye tissues and regulation of osmotic and tensile homeostasis within the eye. Fluid secretion by the ciliary body plays a critical and irreplaceable function in vertebrate vision by providing nutritive support to the cornea and lens, and by maintaining intraocular pressure. Here, we identify TRPV4 (transient receptor potential vanilloid isoform 4) channels as key osmosensors in nonpigmented epithelial (NPE) cells of the mouse ciliary body. Hypotonic swelling and the selective agonist GSK1016790A (EC50 ∼33 nM) induced sustained transmembrane cation currents and cytosolic [Ca2+]i elevations in dissociated and intact NPE cells. Swelling had no effect on [Ca2+]i levels in pigment epithelial (PE) cells, whereas depolarization evoked [Ca2+]i elevations in both NPE and PE cells. Swelling-evoked [Ca2+]i signals were inhibited by the TRPV4 antagonist HC067047 (IC50 ∼0.9 μM) and were absent in Trpv4−/− NPE. In NPE, but not PE, swelling-induced [Ca2+]i signals required phospholipase A2 activation. TRPV4 localization to NPE was confirmed with immunolocalization and excitation mapping approaches, whereas in vivo MRI analysis confirmed TRPV4-mediated signals in the intact mouse ciliary body. Trpv2 and Trpv4 were the most abundant vanilloid transcripts in CB. Overall, our results support a model whereby TRPV4 differentially regulates cell volume, lipid, and calcium signals in NPE and PE cell types and therefore represents a potential target for antiglaucoma medications.

TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye

An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca2+ influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca2+-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension.

Mouse retinal ganglion cell signalling is dynamically modulated through parallel anterograde activation of cannabinoid and vanilloid pathways

Retinal cells use vanilloid transient receptor potential (TRP) channels to integrate light‐evoked signals with ambient mechanical, chemical and temperature information. Localization and function of the polymodal non‐selective cation channel TRPV1 (transient receptor potential vanilloid isoform 1) remains elusive. TRPV1 is expressed in a subset of mouse retinal ganglion cells (RGCs) with peak expression in the mid‐peripheral retina. Endocannabinoids directly activate TRPV1 and inhibit it through cannabinoid type 1 receptors (CB1Rs) and cAMP pathways. Activity‐dependent endocannabinoid release may modulate signal gain in RGCs through simultaneous manipulation of calcium and cAMP signals mediated by TRPV1 and CB1R.

Polymodal TRPV1 and TRPV4 Sensors Colocalize but Do Not Functionally Interact in a Subpopulation of Mouse Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are projection neurons that transmit the visual signal from the retina to the brain. Their excitability and survival can be strongly influenced by mechanical stressors, temperature, lipid metabolites, and inflammatory mediators but the transduction mechanisms for these non-synaptic sensory inputs are not well characterized. Here, we investigate the distribution, functional expression, and localization of two polymodal transducers of mechanical, lipid, and inflammatory signals, TRPV1 and TRPV4 cation channels, in mouse RGCs. The most abundant vanilloid mRNA species was Trpv4, followed by Trpv2 and residual expression of Trpv3 and Trpv1. Immunohistochemical and functional analyses showed that TRPV1 and TRPV4 channels are expressed as separate molecular entities, with TRPV1-only (∼10%), TRPV4-only (∼40%), and TRPV1 + TRPV4 (∼10%) expressing RGC subpopulations. The TRPV1 + TRPV4 cohort included SMI-32-immunopositive alpha RGCs, suggesting potential roles for polymodal signal transduction in modulation of fast visual signaling. Arguing against obligatory heteromerization, optical imaging showed that activation and desensitization of TRPV1 and TRPV4 responses evoked by capsaicin and GSK1016790A are independent of each other. Overall, these data predict that RGC subpopulations will be differentially sensitive to mechanical and inflammatory stressors.